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Burkholderia and application thereof

A technology of Burkholderia bacteria and bacterial liquid, applied in the field of microorganisms, can solve the problems of incapability of citron ketone products, inability to adapt to production, sales and use, etc., and achieves efficient and accurate screening methods, low cost, and fast reaction rate. Effect

Inactive Publication Date: 2021-02-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These chemical synthesis methods use a large number of heavy metal catalysts, which have serious damage to the environment. Although some other non-heavy metal catalysts that can be used will not damage the environment, they all have relatively large safety hazards, and the production efficiency is too low to adapt to production conditions. need
[0004] European food regulations clearly stipulate that citrus ketone products obtained by chemical synthesis cannot be sold and used as a natural flavor, and natural flavors can only be prepared by physical processes (extracted from natural raw materials) or enzyme / microbial processes

Method used

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  • Burkholderia and application thereof
  • Burkholderia and application thereof
  • Burkholderia and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The screening steps for strains with high conversion rate of naringone using valenciene as the sole carbon source are as follows:

[0040] (1) Take a 10g soil sample from the Citrus Engineering Technology Research Center of Hubei Province. Add the soil sample into 90g of sterilized physiological saline and mix well. Take 2 mL of the mixed liquid in 100 mL of minimal medium, and add 1 mL of valenciene (sterilized by 0.22 μm filter membrane, and stored at 4°C), at 37°C, shake at 121r / min for 2 to 7 days, and then use The strains were isolated by the method of gradient dilution and coating, cultured at 37°C until the strains grew completely, and single colonies were picked.

[0041] (2) Preliminary screening: inoculate the picked single colony in LB medium for activation culture. Take an appropriate amount of bacterial liquid in the minimal medium, add 1% valencia citrurene (0.22μm membrane filtration sterilization, store at 4°C), culture at 37°C, 210r / min, measure the O...

Embodiment 2

[0052] Identification of strains

[0053] (1) Morphological and physiological and biochemical assays: Refer to the "Common Bacterial System Identification Manual" to observe the colony morphology of the strains on the solid medium through morphological observation, and at the same time carry out a number of physiological and biochemical indicators of the strains.

[0054] (2) Use a DNA extraction kit to extract bacterial DNA, and amplify 16SrDNA by PCR. The PCR reaction conditions are: 95°C pre-denaturation, 5min; 95°C denaturation, 30sec, 58°C annealing, 30sec, 72°C extension, 45sec, 35sec cycle; 72°C terminal extension, 5min. The PCR amplification products were detected by agarose gel electrophoresis and then sent to Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the known 16SrDNA sequences in GenBank.

Embodiment 3

[0056] Draw the growth curve of the strain in LB medium:

[0057] Inoculate the seed solution of the strain into LB medium at 1% inoculum amount, cultivate at 37°C and 210r / min, use sterile LB medium as a blank control, and measure OD every four hours 600 Value, three parallels, in OD 600 The average value is the ordinate, and the time is the abscissa, and the growth curve of the strain is drawn.

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Abstract

The invention relates to the field of microorganisms, in particular to a Burkholderia and application thereof. A method for preparing the Burkholderia comprises the following steps: firstly, performing preliminary screening, diluting a sample bacterial suspension, coating a flat plate with the diluted sample bacterial suspension, picking a single colony, taking valencene as a unique carbon source,drawing a growth curve for preliminary screening, and selecting a well-growing strain for a next experiment; secondly, performing secondary screening, analyzing and comparing the yield of nootkatonewhich is generated by biological conversion of the valencene through the strain, and selecting a strain with high yield of the nootkatone for a next experiment; and performing streaking on the selected strain, and analyzing and determining a product obtained by the biological conversion of the valencene through the strain and the content of the product. Compared with the conventional physical andchemical synthesis methods, the method for preparing the Burkholderia has the characteristics of high specificity, mild reaction conditions, high reaction rate, high yield, low cost, few reaction steps and the like; moreover, the nootkatone produced in such a way belongs to a natural spice and can be added into foods, cosmetics and other household products.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a Burkholderia bacterium and its application. Background technique [0002] Pomeloone is one of the important substances in the aroma components of pomelo. It has a long-lasting and strong citrus fragrance and is mainly added as a spice in food and cosmetics. Adding a certain amount of narone to cigarettes can increase the aroma of cigarettes, plump the aroma of cigarettes, and reduce irritation; narone has sweet peel and woody aroma, so it can also be used to prepare grapefruit, oranges, tropical fruits, etc. essence. [0003] Natural narone is mainly found in volatile oils such as bergamot oil, grapefruit oil, lemon oil, mandarin oil, lime oil and tangerine oil. However, its natural content in essential oils is generally low, so the actual production and use of narone mainly comes from chemical synthesis. The most common synthetic methods use valenciene as a starting material, ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/26C12R1/01
CPCC12N1/20C12P7/26C12R2001/01C12N1/205
Inventor 任婧楠彭芷芊范刚张璐璐李晓潘思轶
Owner HUAZHONG AGRI UNIV
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